Project summary Amyotrophic lateral sclerosis (ALS) is a predominantly sporadic condition affecting motor neurons (MNs). Its etiology is unknown but several environmental neurotoxicants have been associated with ALS. Though, none have a clear pathogenic role. Only a few of the previous studies investigating the role of environmental factors in ALS have assessed individual biomarkers of exposure (mostly to persistent pollutants) and none has demonstrated a direct concordance between signaling pathways produced by neurotoxic exposure and those implicated in ALS. So far, the progress in this field is hampered by the lack of CNS-relevant specific biomarkers for monitoring both environmental exposure to neurotoxicants and disease progression. Recently, we screened a series of highly prevalent neurotoxicants associated with ALS in vitro and found that mouse and human MNs exhibit a preferential and dose-dependent vulnerability to the metals manganese (Mn) and arsenic (As), and several organophosphate (OP) and pyrethroid (PT) pesticides (e.g. chlorpyrifos [CPS] and cypermethrin). Also, we found that MNs expressing an incompletely penetrant familial ALS variant of TAR DNA-binding protein 43 (G298S TDP-43) are vulnerable to As, Mn and CPS at very low doses that are innocuous to wild-type MNs. TDP-43 pathology is a hallmark of the large majority of ALS cases, both familial and sporadic, so altogether our data suggest that these toxicants could be environmental modifiers of most of the forms of ALS. Intriguingly, excess metals and pathological proteins such as TDP-43 can both be extruded from cells through a homeostatic mechanism that involves the release of tiny membrane-bound compartments called extracellular vesicles (EVs). EVs are now of particular interest as disease biomarkers in other fields. CNS-derived EVs can be isolated from blood because of their ectopic membrane expression of L1CAM. In this study, we will use biospecimens available in the US ALS National Biorepository and from the Target ALS brain bank to reach the following objectives: 1) we will validate hair as a useful biospecimen in ALS for the measure of poorly investigated non-persistent pollutants like OPs and PTs (200 patients x2; taken at 2 ALS stages); 2) we will examine the use of CNS-L1CAM-EVs isolated from the blood of the same 200 patients (2 ALS stages) as a biomarker of environmental exposure (compare metal levels and profiles), as well as a biomarker of disease progression via the accumulation of TDP-43; 3) our epidemiological and statistical analysis will determine association between environmental toxicants, TDP-43 and ALS progression; 4) we will examine concordance between toxicant-specific exposure extracted from gene-expression profiling in mice exposed to CPS and Mn and patient pathogenic transcriptional signatures obtained from our Target ALS consortium to identify disease pathways with therapeutic potential for ALS. By elucidating molecular mechanisms and biomarkers of environmental ALS, our study will have several far-reaching clinical and therapeutic implications.